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The m6A reader HNRNPC promotes non-small cell lung cancer progression by enhancing E2F2 mRNA stability

ABSTRACT: HNRNPC, as one of the m6A reading proteins, has been confirmed to be highly expressed in a variety of tumors and promote the occurrence and progression of tumors. Previous studies have confirmed that HNRNPC significantly promoted the proliferation, migration and invasion of NSCLC cells, and its high expression was related to the clinical stage, lymph node and distant metastasis of patients, and could be used as an indicator of poor prognosis of NSCLC. However, the role of HNRNPC in regulating gene expression through m6A methylation modification in the occurance and progression of NSCLC remains largely unexplored. To investigate the target genes bound by HNRNPC, samples from A549 cells were harvested by RIP lysis buffer, and then the lysates were immunoprecipitated by 5μg anti-HNRNPC antibody. finally, the potential target genes were identified by RIP-seq.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249094 | GEO | 2023/12/05

REPOSITORIES: GEO

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Project description:Endogenous double-stranded RNA is prevented from activating the cytosolic antiviral receptor MDA5 by adenosine-to-inosine editing by ADAR. Consequently, CRISPR/cas9 targeting ADAR in the human monocytic cell line THP-1 induces a spontaneous MDA5-dependent interferon response. This response is synergistically enhanced by concomitant CRISPR/cas9 targeting of hnRNPC. For this study, cas9 transgenic THP-1 were nucleofected with combinations of control gRNA, ADAR gRNA or hnRNPC gRNA to elucidate the basis for synergistic interferon-stmulated gene induction by combined ADAR and hnRNPC targeting. As a control for interferon-stimulated gene induction, IFN-alpha treated THP-1 were included, as well. To investigate potential interferon inducing mechanisms, we quantified gene expression, intron expression as well as the expression of regions rich in adenosine-to-inosine editing clusters. In a second part, nuclear vs cytosolic distribution of RNA rich in adenosine-to-inosine clusters was investigated by sequencing of total RNA or RNA from cytosolic and nuclear extracts of ADAR and/or hnRNPC targeted cells. In the first part of the study, STING-deficient, cas9 transgenic THP-1 were nucleofected with combinations of control, GFP-targeting, ADAR-targeting or hnRNPC-targeting gRNA and cells collected for total RNA extraction on days three, four and five after nucleofection. In addition, another control-nucleofected sample was treated on day 3 for 24 h. The experiment was repeated three times leading to three replicates per condition. In the second part, WT cas9-transgenic THP-1 were nucleofected with combinations of control, ADAR-targeting or hnRNPC-targeting gRNA and cells were either collected for total RNA extraction or separated into digitonin-soluble (cytosolic) and digitonin-resistant (nuclear and mitochondrial) compartments and then RNA extracted. The experiment was repeated twice leading to two replicates per condition. Differential gene expression analysis confirmed synergistic induction of interferon-stimulated genes. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters or regions enriched in editing clusters (ECRs) in general. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. Part 2 of the study confirmed cytosolic access of a subset of ECRs to the cytosol.

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The m6A reader HNRNPC promotes non-small cell lung cancer progression by enhancing E2F2 mRNA stability

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